Doxorubicin (DXR) is a potent chemotherapeutic agent effective against a broad spectrum of neoplasms (Aubel-Sadron et al and Young}. However, clinical use of the drug in free form is limited by serious side effects and acute toxicity including malaise, nausea, vomiting myelosuppression, and severe alopecia. In addition, cumulative and irreversible cardiac damage occurs with repeated administration, which seriously limits the use of the drug in protracted treatment (Young).
One approach which has been used to reduce DXR toxicity in vivo is to administer the drug in liposome-entrapped form. Animal studies and, more recently, clinical testing demonstrate that DXR in liposome-bound form retains its therapeutic effectiveness against animal tumors, but is significantly less toxic, as judged by reduced mortality and reduction in cardiotixic effects (Forssen, Gabizon 1985). The drug-protective effect of liposomes is due, at least in part, to a marked alteration in tissue disposition and drug-release rate of the injected drug (Gabizon 1982; Gabizon 1983; Juliano).
Recent animal model studies point to three factors which are important in achieving increased therapeutic action and reduced toxicity in DXR/liposomes. One of these is liposome size. Studies aimed at determining the biodistribution and drug clearance of DXR/liposomes after intravenous administration, as a function of liposome size, have been conducted (Gabizon 1982, 1983, and U.S. Pat. application for "Liposome/Anthraquinone Drug Composition and Method, Ser. No. 806,084, filed Dec. 6, 1985, now U.S. Pat. No. 4,797,285). Briefly summarizing the results, DXR/liposomes having average sizes of between about 0.1-0.2 show increased drug levels in the liver and spleen, and decreased drug levels in heart, lung, intestine, kidney, and skeletal muscles when compared with the free drug. Liposomes of this size are thus particularly advantageous in treating liver- and spleen-localized tumors and in reducing toxicity related to drug levels in non-target tissues, particularly the heart. Liposomes in this size range also show slower drug clearance in liver and spleen tissue. The biodistribution and drug clearance rates in small unilamellar vesicles (0.03-0.08 size range approximately) was intermediate that of the larger liposomes and free drug.
For a variety of reasons, the optimal upper size limit of the liposomes is about 0.5 microns and, preferably, about 0.2-0.3 microns. First, desired target tissue regions, such as liver sinusoids and parenchyma, spleen, and bone marrow are more accessible to liposomes smaller than about 0.2-0.3 microns. Secondly, liposomes in the 0.2-0.3 micron size range can be readily sterilized by filtration through a depth filter. Vesicles of this size also show little tendency to aggregate on storage, thus reducing a potentially serious problem when the composition is administered parenterally. Finally, liposomes which have been sized down to the submicron range show more uniform biodistribution and drug clearance characteristics, since they have more uniform sizes.
A second feature of DXR/liposomes which is important to therapeutic effectiveness is the extent of chemical degradation of lipid and drug components which can occur on storage. Phospholipid degradation can take the form of hydrolytic release of fatty acyl chain groups and lipid peroxidative damage, particularly at unsaturated bond regions in lipid acyl chain moieties (Gutterage, 1984; Sunamoto). In addition, anthaquinone-type drugs, such as DXR, are themselves capable of initiating oxidative reactions (Goormaghtigh, 1984; Gutteridge, 1984a), and in the presence of lipids, appear to contribute to free radical/oxidative reactions, and also undergo rapid chemical changes on storage in liposomes, as reported in the above-cited patent application for "Liposome/Anthraquinone Drug Composition and Method".
In studies carried out in support of the just-cited application, it was found that the combined presence of a lipid-soluble free radical quencher, such as alpha-tocopheral, and a water-soluble trihydroxamic chelating agent, such as desferal, reduced peroxidative damage to lipid and drug components substantially more than the sum of individual protection provided by either protective agent alone. A key feature of this protective effect appears to involve chelation of ferric iron in a form which does not readily catalyze peroxidation formation, combined with lipid-phase free-radical quenching by the lipophilic quencher.
The toxicity of DXR/liposome formulations is also sensitive to the percentage of free drug in the formulation. Clinical trial evaluations indicate that DXR/liposomes containing moderate levels of free drug (about 35%) produce substantially greater toxicity at a 50mg/m.sup.2 dose than do DXR/liposomes with low free drug (10-15%) administered at a 70 mg/m.sup.2 dose. There are a variety of methods available for removing free DXR from DXR/liposomes preparations, including centrifugation, diafiltration, filtration by molecular sieve chromatography, and, as disclosed in U.S. Pat. No. 4,460,577, by filtration through an ion-exchange resin. These methods are effective in reducing free drug levels in freshly prepared DXR/liposome preparations to 10% or less of total DXR. However, on storage in solution, free drug may be gradually released as increased lipid and drug oxidative damage occurs. Conventional lyophilization/and reconstitution methods for storing DXR/liposomes in a dried state lead to substantial release of free drug on reconstitution. Conventional lyophilization and reconstitution procedures result in a substantial release of liposome-associated DXR, typically resulting in 20-30% free drug in the reconstituted liposome suspension.